Abstract 150: FAK/Pyk2 Inhibitor Prevents Mitochondrial Ca 2 + Overload and Cardiac Injury during Adrenergic Stimulation.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Jin O-Uchi ◽  
Bong Sook Jhun ◽  
Stephen Hurst ◽  
Shey-Shing Sheu
Keyword(s):  
Heart Failure ◽  
Cell Injury ◽  
Mitochondrial Permeability Transition ◽  
Mitochondrial Protein ◽  
Cardiac Injury ◽  
Dominant Negative ◽  
Ros Generation ◽  
Adrenergic Stimulation ◽  
Human Heart Failure ◽  
Tyrosine Kinase 2

Introduction: Proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) are abundantly expressed in cancer cells. In addition, these kinases are the potent therapeutic targets for cancer treatment and currently several selective FAK/Pyk2 inhibitors are in clinical trials. Recent study revealed that Pyk2 is also highly expressed in heart tissue and significantly activated during human heart failure. We have recently reported that α 1 -adrenoceptor (α 1 -AR) stimulation accelerates mitochondrial Ca 2+ uptake through Pyk2-dependent phospholylation of mitochondrial Ca 2+ uniporter (MCU). However, the roles of Pyk2 in cardiac mitochondrial physiology and pathophysiology have not been well established. Hypothesis: Persistent adrenergic signaling activates cell death signaling via Pyk2-dependent MCU activation and mitochondrial Ca 2+ overload. Methods: Using H9C2 cardiac myoblasts, mitochondrial Ca 2+ and reactive oxygen species (ROS) were measured using mitochondrial matrix-targeted Ca 2+ -sensitive inverse pericam and MitoSOX, respectively. Mitochondrial permeability transition pore (mPTP) activity was observed by measuring the amount of cytochrome c in cytosol by Western blotting or by monitoring the release of GFP-tagged mitochondrial protein, Smac-GFP using confocal microscopy. Results: Pyk2 was not only expressed in cytosol, but also in cardiac mitochondria. α 1 -AR agonist phenylephrine activated mitochondrial Pyk2 and enhanced mitochondrial Ca 2+ uptake via Pyk2-dependent MCU phosphorylation. In addition, persistent α 1 -AR stimulation increases ROS, activity of mPTP. These effects were abolished by co-expression of dominant-negative MCU or kinase-dead Pyk2, suggesting that Pyk2-dependent MCU activation followed by mitochondrial Ca 2+ overload are critical for this mechanism. Moreover, pretreatment of a potent FAK/Pyk2 inhibitor PF-431396 also effectively inhibited α 1 -AR-mediated ROS generation and mPTP activation. Conclusion: FAK/Pyk2 inhibitor prevents mitochondrial Ca 2+ overload, oxidative stress and mitochondrial injury under persistent adrenergic stimulation. Thus, Pyk2 may become a novel potent therapeutic target for preventing cardiac cell injury and death during heart failure.

scholarly journals Catecholamine Surges Cause Cardiomyocyte Necroptosis via a RIPK1–RIPK3-Dependent Pathway in Mice

2021 ◽  
Vol 8 ◽  
Author(s):  
Penglong Wu ◽  
Mingqi Cai ◽  
Jinbao Liu ◽  
Xuejun Wang
Keyword(s):  
Heart Failure ◽  
Cardiac Injury ◽  
Underlying Mechanism ◽  
Selective Inhibitor ◽  
Blue Dye ◽  
Wild Type ◽  
Adrenergic Stimulation ◽  
Protein Levels ◽  
Regulated Necrosis ◽  
Dependent Pathway

Background: Catecholamine surges and resultant excessive β-adrenergic stimulation occur in a broad spectrum of diseases. Excessive β-adrenergic stimulation causes cardiomyocyte necrosis, but the underlying mechanism remains obscure. Necroptosis, a major form of regulated necrosis mediated by RIPK3-centered pathways, is implicated in heart failure; however, it remains unknown whether excessive β-adrenergic stimulation-induced cardiac injury involves necroptosis. Hence, we conducted the present study to address these critical gaps.Methods and Results: Two consecutive daily injections of isoproterenol (ISO; 85 mg/kg, s.c.) or saline were administered to adult mixed-sex mice. At 24 h after the second ISO injection, cardiac area with Evans blue dye (EBD) uptake and myocardial protein levels of CD45, RIPK1, Ser166-phosphorylated RIPK1, RIPK3, and Ser345-phosphorylated MLKL (p-MLKL) were significantly greater, while Ser321-phosphorylated RIPK1 was significantly lower, in the ISO-treated than in saline-treated wild-type (WT) mice. The ISO-induced increase of EBD uptake was markedly less in RIPK3−/− mice compared with WT mice (p = 0.016). Pretreatment with the RIPK1-selective inhibitor necrostatin-1 diminished ISO-induced increases in RIPK3 and p-MLKL in WT mice and significantly attenuated ISO-induced increases of EBD uptake in WT but not RIPK3−/− mice.Conclusions: A large proportion of cardiomyocyte necrosis induced by excessive β-adrenergic stimulation belongs to necroptosis and is mediated by a RIPK1–RIPK3-dependent pathway, identifying RIPK1 and RIPK3 as potential therapeutic targets for catecholamine surges.

ASSOCIATION OF HUMANIN A NOVEL MITOCHONDRIAL PROTEIN WITH HUMAN HEART FAILURE: AN ASSESSMENT OF MORTALITY AND CLINICAL BIOMARKERS

2019 ◽  
Vol 73 (9) ◽  
pp. 1023 ◽  
Author(s):  
Sati Patel ◽  
Jennifer D. Hwang ◽  
Hemalatha Narayanasamy ◽  
Luanda Grazette ◽  
Jeffrey Tran ◽  
...  
Keyword(s):  
Heart Failure ◽  
Human Heart ◽  
Mitochondrial Protein ◽  
Human Heart Failure ◽  
Clinical Biomarkers

scholarly journals Na+/Ca2+ exchanger overexpression impairs frequency- and ouabain-dependent cell shortening in adult rat cardiomyocytes

2004 ◽  
Vol 287 (4) ◽  
pp. H1435-H1445 ◽  
Author(s):  
Birgit Bölck ◽  
Götz Münch ◽  
Peter Mackenstein ◽  
Martin Hellmich ◽  
Ingo Hirsch ◽  
...  
Keyword(s):  
Heart Failure ◽  
Gene Transfer ◽  
Mode Of Action ◽  
Positive Inotropic Effect ◽  
Inotropic Effect ◽  
Adrenergic Stimulation ◽  
Human Heart Failure ◽  
Rat Cardiomyocytes ◽  
Cell Shortening ◽  
Reverse Mode

The Na+/Ca2+ exchanger (NCX) may influence cardiac function depending on its predominant mode of action, forward mode or reverse mode, during the contraction-relaxation cycle. The intracellular Na+ concentration ([Na+]i) and the duration of the action potential as well as the level of NCX protein expression regulate the mode of action of NCX. [Na+]i and NCX expression have been reported to be increased in human heart failure. Nevertheless, the consequences of altered NCX expression in heart failure are still a matter of discussion. We aimed to characterize the influence of NCX expression on intracellular Ca2+ transport in rat cardiomyocytes by adenoviral-mediated gene transfer. A five- to ninefold (dose dependent) overexpression of NCX protein was achieved after 48 h by somatic gene transfer (Ad.NCX.GFP) versus control (Ad.GFP). NCX activity, determined by Na+ gradient-dependent 45Ca2+-uptake, was significantly increased. The protein expressions of sarco(endo)plasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin were unaffected by NCX overexpression. Fractional shortening (FS) of isolated cardiomyocytes was significantly increased at low stimulation rates in Ad.NCX.GFP. After a step-wise enhancing frequency of stimulation to 3.0 Hz, FS remained unaffected in Ad.GFP cells but declined in Ad.NCX.GFP cells. The positive inotropic effect of the cardiac glycoside ouabain was less effective in Ad.NCX.GFP cells, whereas the positive inotropic effect of β-adrenergic stimulation remained unchanged. In conclusion, NCX overexpression results in a reduced cell shortening at higher stimulation frequencies as well as after inhibition of sarcolemmal Na+-K+-ATPase, i.e., in conditions with enhanced [Na+]i. At low stimulation rates, increased NCX expression enhances both intracellular systolic Ca2+ and contraction amplitude.

Abstract 12646: Oxidation-Dependent Phosphomimetic Effect of a Human Heart Failure Mutation of Phospholamban

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Neha Abrol ◽  
Pieter P de Tombe ◽  
Seth L Robia
Keyword(s):  
Heart Failure ◽  
Molecular Mechanism ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Premature Death ◽  
Oxidative Modification ◽  
Live Cells ◽  
Adrenergic Stimulation ◽  
Cysteine Oxidation ◽  
Human Heart Failure

Rationale: A naturally-occurring, missense Arg9-to-Cys (R9C) mutation of phospholamban (PLB) triggers cardiomyopathy and premature death in humans. However, the fundamental molecular mechanism underlying the cardiotoxic role of R9C-PLB in sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) regulation and cardiomyocyte Ca 2+ handling is not clear. Objective: The goal of this study was to investigate the acute physiological consequences of R9C-PLB mutation on cardiomyocyte Ca 2+ kinetics and contractility and identify the molecular mechanism underlying R9C pathology. Methods and Results: We measured the physiological consequences of R9C-PLB mutation on Ca 2+ transients and sarcomere shortening in adult cardiomyocytes at increasing pacing frequencies. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we fused fluorescent protein tags to PLB and SERCA, and compared the effect of R9C and pentamer-destabilizing mutation (SSS) on PLB oligomerization and PLB-SERCA interaction. Fluorescence resonance energy transfer (FRET) measurements in live cells revealed that R9C exhibited an increased affinity of PLB oligomerization, and a decreased binding affinity to SERCA due to an oxidative modification which mimics phosphorylation. Real-time FRET analysis in cardiomyocytes revealed that R9C-PLB exhibits enhanced sensitivity to oxidative stress, which is a prevailing condition in heart failure. Conclusions: We conclude that the primary mechanism of R9C pathology is a phosphomimetic effect of PLB cysteine oxidation, manifested as increased oligomerization and a change in the structure of the PLB-SERCA regulatory complex.

Abstract 359: Higher ROS Generation and Lower Threshold of mPTP Opening May Underlie Increased Vulnerability of Late Pregnant Heart to Ischemia-Reperfusion Injury

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Jingyuan Li ◽  
Andrea Iorga ◽  
Ji-Youn Youn ◽  
Hua Cai ◽  
Vera Regitz-Zagrosek ◽  
...  
Keyword(s):  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Mitochondrial Protein ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Ros Generation ◽  
Hemodynamic Parameters ◽  
Mptp Opening

Although the murine late pregnant (LP) heart is speculated to be a better functioning heart during physiological conditions, the susceptibility of LP hearts to I/R injury is still unknown. The aims of this study were to investigate the cardiac vulnerability of LP rodents to ischemia/reperfusion (I/R) injury and to explore its underlying mechanisms. In-vivo female rat hearts (non-pregnant (NP) or LP) or Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion. The infarct size was ∼4 fold larger in LP compared to NP both in the in-vivo rat model and ex-vivo mouse model. The hemodynamic parameters were similar between NP and LP before ischemia. However, the postischemic functional recovery was extremely poor in LP mice comparing to NP mice. RPP was reduced from 12818±1213mmHg*beats/min in NP to 1617± 287mmHg*beats/min in LP mice at the end of reperfusion. Interestingly, all of the hemodynamic parameters almost fully recovered in hearts seven days post-partum (PP7)( RPP= 9604±1215 mmHg*beats/min). To explore the mitochondrial function involvement in the higher vulnerability of LP hearts to I/R injury, mitochondrial respiration and ROS production were measured. Respiratory control index(RCI) were significantly decreased in LP subjected to I/R compared to NP and PP7 (RCI=1.9±0.1 in LP, 4.0±0.5 in NP and 3.9±0.5 in PP7, P<0.05 LP vs. NP and PP7). The superoxide production was also significantly higher in isolated cardiac mitochondria from LP hearts subjected to I/R injury (10.7±1.7mM/min/mg protein in NP; 21.3±3.1mM/min/mg protein in LP and 9.3±3.3mM/min/mg protein in PP7; p<0.05 LP vs. NP and PP7). The threshold for opening of mitochondrial permeability transition pore (mPTP) in response to Ca2+ overload was much lower in LP hearts (calcium retention capacity(CRC)=167±10 nmol/mg-mitochondrial protein) compared with NP (233±18 nmol/mg-mitochondrial protein) and PP7 (260±12 nmol/mg-mitochondrial protein, P<0.01). In conclusion, the higher susceptibility of LP hearts to I/R injury is associated with a lower threshold for triggering the mitochondrial permeability transition pore (mPTP) opening in response to Ca2+ overload which may at least be in part due to higher ROS generation and lower mitochondrial respiration.

Abstract 1240: Sodium Channel Splice Variants Seen in Human Heart Failure Result in Dominant Negative Suppression of the Wild-Type Current

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Lijuan L Shang ◽  
Shamarendra Sanyal ◽  
Samuel C Dudley
Keyword(s):  
Heart Failure ◽  
Cell Line ◽  
Human Heart ◽  
Splice Variants ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Human Heart Failure ◽  
Mrna Splice ◽  
Hek Cells

Patients with heart failure have increased risk of death and reduced Na + channels. We have identified a possible mechanism whereby Na + channels are decreased in human heart failure. We have observed that heart failure is associated with increases of 14.2 fold and 3.8 fold in two (C and D) of three cardiac Na + channel mRNA splice variants. These variants encode for channels that are truncated before the domain IV pore-forming loop and are predicted to be non-functional. We tested this prediction by expressing the truncated forms in HEK cells. The truncated SCN5A cDNAs labeled either with IRES (internal ribosome entry site)-mediated GFP or fused GFP were transitively transfected into HEK cells or a HEK cell line stably expressing the native human SCN5A. HEK cell lines stably expressing the truncation variants were created by selection for three weeks with geneticin after transfection. Neither of the two truncations generated current when transfected alone. When transfected in a cell line stably expressing wild-type Na + channels, the presence of the truncations resulted in 54.6% (±8.5, p<0.01, N=14) and 56.0% (±8.9, p<0.01, N=10) reductions in peak current with the C and D variants respectively when compared to the wild-type alone. The reduction of wild-type current in the presence of the truncations showed variant dose-dependency when varying the amount of vector encoding for the splice variants. Laser fluorescent scanning for N-terminal GFP labeled Na + channels confirmed a reduced number of wild-type channels in the membrane in the presence of mRNA for the two truncations. In conclusion, truncated Na + channel mRNA splice variants are increased in human heart failure. These truncations have a dominant negative effect on the wild-type allele. This may help explain the reduced Na + current and increased arrhythmic risk in heart failure.

Abstract 093: Adrenergic Stimulation Induces Mitochondrial Fragmentation and Cell Injury through PKD1-dependent Phosphorylation of DLP1 in H9c2 Cardiac Myoblasts.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Bong Sook Jhun ◽  
Jin O-Uchi ◽  
Stephen Hurst ◽  
Shey-Shing Sheu
Keyword(s):  
Protein Kinase ◽  
Mitochondrial Dysfunction ◽  
Cell Injury ◽  
Mitochondrial Fission ◽  
Dominant Negative ◽  
Mitochondrial Morphology ◽  
Catecholamine Level ◽  
Adrenergic Stimulation ◽  
Mitochondrial Fragmentation ◽  
Cardiac Myoblasts

Introduction: Regulation of mitochondrial morphology and dynamics is crucial for the maintenance of various cellular functions in cardiac myocytes. Abnormal mitochondrial morphologies concomitant with mitochondrial dysfunction are frequently observed in various pathophysiological states of human heart such as heart failure, where the catecholamine level is elevated. However, it is still unclear what kinds of cardiac signaling pathways regulate mitochondrial morphology and function under pathophysiological conditions. Hypothesis: Adrenergic signaling induces cardiac mitochondrial morphology changes and mitochondrial dysfunction, which simultaneously contribute to cardiac injury. Methods: H9c2 cardiac myoblasts were stimulated by α 1 -adrenoceptor (α 1 -AR) agonist phenylephrine and mitochondrial morphology was monitored by confocal microscopy. Translocation and phosphorylation of a mitochondrial fission protein, dynamin-like protein 1 (DLP1) was observed from whole cell lysates, cytosolic proteins and mitochondrial proteins by western blotting. Results: We found that persistent α 1 -AR stimulation induced mitochondrial fragmentation, followed by an increase in the production of mitochondrial reactive oxygen species (ROS) and the release of cytochrome c from mitochondria to the cytosol in H9c2 cardiac myoblasts. These effects were abolished by the treatment of α 1 -AR antagonist, prazosin. Further, mitochondrial fragmentation by α 1 -AR stimulation was inhibited by expression of the dominant-negative fission mutant DLP1-K38A, suggesting that the mitochondrial fission is required for mitochondrial fragmentation observed in α 1 -AR stimulation. We also found that DLP1 was translocated from cytosol to mitochondria under α 1 -AR stimulation. In addition, activation of protein kinase D1 (PKD1), a protein kinase downstream of α 1 -AR signaling, led to the phosphorylation of DLP1 at serine 637 which lies within a putative PKD phosphorylation consensus motif. Conclusion: α 1 -AR signaling induces mitochondrial fragmentation and cell injury, possibly through PKD1-dependent phosphorylation of DLP1.

Abstract 5361: PDK1 Plays Crucial Roles in β-Adrenergic Response and Cell Survival in the Heart

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Kaoru Ito ◽  
Hiroshi Akazawa ◽  
Noritaka Yasuda ◽  
Haruaki Nakaya ◽  
Issei Komuro
Keyword(s):  
Heart Failure ◽  
Cardiac Function ◽  
Gene Disruption ◽  
Systolic Function ◽  
Severe Heart Failure ◽  
Left Ventricular ◽  
Cardiomyocyte Apoptosis ◽  
Adrenergic Stimulation ◽  
Human Heart Failure ◽  
Bax Protein

Background: The 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a homeostatic role in the regulation of cellular function in multiple organs, by working downstream of phosphatidylinositol-3 kinase (PI3-K) through activating several kinases including Akt and p70 S6 kinase. We found that myocardial expression of PDK1 was decreased in murine models of heart failure. Although previous studies have reported that PDK1 is important for the regulation of cardiomyocyte size, the precise role of PDK1 in the heart remains to be fully characterized. Methods and Results: To elucidate the roles of PDK1 in the postnatal heart, we generated tamoxifen-inducible heart specific PDK1 knockout (PDK1-KO) mice. Deletion of PDK1 at the age of 10 weeks caused severe heart failure. At 1 week after Pdk1 gene disruption, left ventricular systolic function was already deteriorated without reduction in cardiomyocyte size. Langendorff-perfused hearts from PDK1-KO mice exhibited impaired responsiveness to isoproterenol in spite of preserved responsiveness to forskolin. In PDK1-KO hearts, PI3-K γ activity was enhanced and the expression levels of β1-adrenergic receptor (β1AR) in membrane fraction were decreased. Overexpression of PIK-domain of PI3-K γ blocked β1AR internalization by competitively inhibiting the association between PI3-K γ and G-protein coupled receptor kinase 2, and improved cardiac function in PDK1-KO hearts. In addition, we found that cardiomyocyte apoptosis was significantly increased 1 week after Pdk1 gene disruption. PDK1-KO hearts showed reduction of Akt and SGK activities, upregulation of Bax protein and endoplasmic reticulum stress signals, which were the potential causes of cardiomyocyte apoptosis. Overexpression of Bcl-2 in PDK1-KO hearts prevented cardiomyocyte apoptosis and partially improved cardiac function in PDK1-KO mice. Conclusions : These results suggest that PDK1 is essential for normal cardiac function by not only preserving responsiveness to β-adrenergic stimulation but also preventing cardiomyocyte apoptosis. Since PDK1-KO mice reproduces many aspects of human heart failure, we propose that PDK1 may be a promising molecule targeted for the treatment of heart failure.

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